title: Site-directed mutagenesis reveals functional contribution of Thr218, Lys220 and Asp304 in chymosin.
abstract: The functional contributions of amino acid residues Thr218 and Asp304 of chymosin, both of which are highly conserved in the aspartic proteinases, are analysed by means of site-directed mutagenesis. The optimum pH values, milk-clotting (C) and proteolytic (P) activities and kinetic parameters for synthetic oligopeptides as substrates were examined for the mutant enzymes. The mutation Thr218Ser caused a marked increase in the C/P ratio, which seemed to be due to a change in substrate recognition. Although the negative charge of Asp304 had been expected to play a role in lowering the optimum pH values in the aspartic proteinases, this turned out not to be the case in chymosin because both the mutations Asp304Ala and Asp304Glu caused a similar shift of the optimum pH towards the acidic side. In addition, the mutation Lys220Leu, which we generated previously, was found to cause a decrease in the C/P ratio, mainly due to the increase in the proteolytic activity.
authors.affiliation: Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Suzuki
authors.forename: A
authors.initials: A
authors.lastname: Hamu
authors.forename: M
authors.initials: M
authors.lastname: Nishiyama
authors.forename: S
authors.initials: S
authors.lastname: Horinouchi
authors.forename: T
authors.initials: T
authors.lastname: Beppu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D012085:Chymosin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D012085:Chymosin